During transcription, a single-stranded RNA complementary to the DNA sequence to be transcribed is formed by the action of RNA polymerases. Initiation of transcription in eucaryotic cells is regulated by complex interactions between cis-acting DNA motifs, and trans-acting protein factors. Among the cis-acting regulatory regions are sequences of DNA, termed promoters. A promoter is located close to the transcription initiation site and comprises a nucleotide sequence that associates with an RNA polymerase, either directly or indirectly. Promoters usually consist of proximal (e.g. TATA box) and more distant elements (e.g. CCAAT box). Enhancers are cis-acting DNA motifs which may be situated 5-prime and/or 3-prime from the initiation site.
Both promoters and enhancers are generally composed of several discrete, often redundant, elements each of which may be recognized by one or more trans-acting regulatory proteins, known as transcription factors. Regulation of the complex patterns of gene expression observed both spatially and temporally, in all developing organisms, is thought to arise from the interaction of enhancer- and promoter-bound, general and tissue-preferred transcription factors with DNA (Izawa T, Foster R and Chua N H, 1993, J. Mol. Biol. 230:1131-1144; Menkens A E, Schindler U and Cashmore A R, 1995, Trends in Biochem Sci 13:506-510). Developmental decisions in organisms as diverse as Drosophila melanogaster, Saccaromyces cerevisiae, Arabidopsis thaliana and Pinus radiata are regulated by transcription factors. These DNA-binding regulatory molecules have been shown to control the expression of genes responsible for the differentiation of different cell types, for example, the differentiation of leaf trichomes and xylem tissue in Arabidopsis thaliana (Kirik V, Schnittger A, Radchuk V, Adler K, Hulskamp M and Baumlein H, 2001, Dev Biol. 235(2):366-77, Baima S, Possenti M, Matteucci A, Wisman E, Altamura M M, Ruberti I and Morelli G., 2001 Plant Physiol. 126(2):643-55, formation of endoderm from embryonic cells in Xenopus laevis and the initiation of gene expression in response to environmental and phytohormonal stress in plants (Yanagisawa S and Sheen J, 1998, The Plant Cell 10:75-89).
Transcription factors generally bind DNA in a sequence-specific manner and either activate or repress transcription initiation. The specific mechanisms of these interactions remain to be fully elucidated. At least three types of separate domains have been identified within transcription factors. One is essential for sequence-specific DNA recognition, one for the activation/repression of transcriptional initiation, and one for the formation of protein-protein interactions (such as dimerization). Studies indicate that many plant transcription factors can be grouped into distinct classes based on their conserved DNA binding domains (Katagiri F and Chua N H, 1992, Trends Genet. 8:22-27; Menkens A E, Schindler U and Cashmore A R, 1995, Trends in Biochem Sci. 13:506-510; Martin C and Paz-Ares J, 1997, Trends Genet. 13:67-73). Each member of these families interacts and binds with distinct DNA sequence motifs that are often found in multiple gene promoters controlled by different regulatory signals.
Several transcription factor families have been identified in plants. For example, nucleotide sequences encoding the following transcription factors families have been identified: Alfin-like, AP2 (APETALA2) and EREBPs (ethylene-responsive element binding proteins), ARF, AUX/IAA, bHLH, bZIP, C2C2 (Zn), C2C2 (Co-like), C2C2 (Dof), C2C2 (GATA), C2C2 (YABBY), C2H2 (Zn), C3H-type, CCAAT, CCAAT HAP3, CCAAT HAP5, CPP (Zn), DRAP1, E2F/DP, GARP, GRAS, HMG-BOX, HOMEO BOX, HSF, Jumanji, LFY, LIM, MADS Box (SEQ ID NO: 3668), MYB, NAC, NIN-like, Polycomb-like, RAV-like, SBP, TCP, TFIID, Transfactor, Trihelix, TUBBY, and WRKY (SEQ ID NO: 3670).
Because transcription factors regulate transcription and orchestrate gene expression in plants and other organisms, control of transcription factor gene expression provides a powerful means for altering plant phenotype. The multigenic control of plant phenotype presents difficulties in determining the genes responsible for phenotypic determination. One major obstacle to identifying genes and gene expression differences that contribute to phenotype in plants is the difficulty with which the expression of more than a handful of genes can be studied concurrently. Another difficulty in identifying and understanding gene expression and the interrelationship of the genes that contribute to plant phenotype is the high degree of sensitivity to environmental factors that plants demonstrate.
There have been recent advances using genome-wide expression profiling. In particular, the use of DNA microarrays has been useful to examine the expression of a large number of genes in a single experiment. Several studies of plant gene responses to developmental and environmental stimuli have been conducted using expression profiling. For example, microarray analysis was employed to study gene expression during fruit ripening in strawberry, Aharoni et al., Plant Physiol. 129:1019-1031 (2002), wound response in Arabodopsis, Cheong et al., Plant Physiol. 129:661-7 (2002), pathogen response in Arabodopsis, Schenk et al., Proc. Nat'l Acad. Sci. 97:11655-60 (2000), and auxin response in soybean, Thibaud-Nissen et al., Plant Physiol. 132:118. Whetten et al., Plant Mol. Biol. 47:275-91 (2001) discloses expression profiling of cell wall biosynthetic genes in Pinus taeda L. using cDNA probes. Whetten et al. examined genes which were differentially expressed between differentiating juvenile and mature secondary xylem. Additionally, to determine the effect of certain environmental stimuli on gene expression, gene expression in compression wood was compared to normal wood. A total of 156 of the 2300 elements examined showed differential expression. Whetten, supra at 285. Comparison of juvenile wood to mature wood showed 188 elements as differentially expressed. Id. at 286.
Although expression profiling and, in particular, DNA microarrays provide a convenient tool for genome-wide expression analysis, their use has been limited to organisms for which the complete genome sequence or a large cDNA collection is available. See Hertzberg et al., Proc. Nat'l Acad. Sci. 98:14732-7 (2001a), Hertzberg et al., Plant J., 25:585 (2001b). For example, Whetten, supra, states, “A more complete analysis of this interesting question awaits the completion of a larger set of both pine and poplar ESTs.” Whetten et al. at 286. Furthermore, microarrays comprising cDNA or EST probes may not be able to distinguish genes of the same family because of sequence similarities among the genes. That is, cDNAs or ESTs, when used as microarray probes, may bind to more than one gene of the same family.
Methods of manipulating gene expression to yield a plant with a more desirable phenotype would be facilitated by a better understanding of transcription factor gene expression in various types of plant tissue, at different stages of plant development, and upon stimulation by different environmental cues. The ability to control plant architecture and agronomically important traits would be improved by a better understanding of how cell cycle gene expression effects formation of plant tissues, how cell cycle gene expression causes plant cells to enter or exit cell division, and how plant growth and transcription factor gene are connected. Among the large number of transcription factor genes, the expression of which can change during development of a plant, only a fraction are likely to effect phenotype.
Accordingly, there exists a need for transcription factors that can be used for regulating gene expression in plants.